[47] Purification and characterization of mammalian and chicken ferrochelatase

Dailey, Harry A.; Fleming, J E; Harbin, Bertille M.

doi:10.1016/s0076-6879(86)23049-9

Cited by 43 publications

(23 citation statements)

References 13 publications

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“…Ferrochelatase (FC), 1 the last enzyme of the heme biosynthesis pathway, is located on the matrix side of the inner mitochondrial membrane and catalyzes the insertion of ferrous iron into protoporphyrin to form heme (16). FC protein has been purified from several mammalian species, and in all has a molecular size of 40-42 kD (17). However, radiation inactivation has demonstrated that the functional size of FC in bovine liver mitochondria is ‫ف‬ 80 kD (18), suggesting that the enzyme exists as a homodimer in the mitochondrial membrane.…”

Section: Introductionmentioning

confidence: 99%

Molecular defects in ferrochelatase in patients with protoporphyria requiring liver transplantation.

Bloomer¹,

Bruzzone²,

Zhu³

et al. 1998

J. Clin. Invest.

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Protoporphyria is a genetic disorder in which a deficiency of mitochondrial ferrochelatase activity causes accumulation of protoporphyrin that produces severe liver damage in some patients. In this study, mutations of the ferrochelatase gene were examined in eight unrelated patients who had liver transplantation. RNA was prepared from liver and/ or lymphoblasts, and specific reverse transcriptase-nested polymerase chain reactions amplified and sequenced ferrochelatase cDNAs. Products shorter than normal resulted from an exon 3 deletion in three patients, exon 10 deletion in two, exon 2 deletion in one, and deletion of five nucleotides in exon 5 in one. Sequence of normal-size products revealed no other mutations. Western blot showed a reduced quantity of normal-size ferrochelatase protein in protoporphyria liver compared with normal liver (19-51%, mean 32% of normal). Levels of the mitochondrial protein F 1 -ATPase ␤ -subunit were not decreased to a similar degree. Liver ferrochelatase activity was reduced more than could be explained by the decrease in ferrochelatase protein (4-20%, mean 9% of normal). These results establish genetic heterogeneity in the most severe phenotype of protoporphyria. However, the gene mutations found share the property of causing a major structural alteration in the ferrochelatase protein.

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Section: Introductionmentioning

confidence: 99%

Molecular defects in ferrochelatase in patients with protoporphyria requiring liver transplantation.

Bloomer¹,

Bruzzone²,

Zhu³

et al. 1998

J. Clin. Invest.

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“…The latter process is catalyzed by enzymes, and a model for the catalytic function of the enzyme has been proposed as the porphyrin distortion caused by steric and electronic interactions with amino acid residues at the enzyme active site. [10][11][12][13][14][15][16] For example, the metalloporphyrin formation is a key reaction of the biosynthesis of heme, since the final step of the biosynthesis is the Fe(II) ion incorporation in vivo into the protoporphyrin IX. [17][18][19][20] For an understanding of the metalloporphyrin formation mechanism and the role of the enzymatic catalysis, further kinetic studies of the metalation have been required.…”

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confidence: 99%

Dynamic Study of Metal-Ion Incorporation into Porphyrins Based on the Dynamic Characterization of Metal Ions and on Sitting-Atop Complex Formation

2001

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The metalloporphyrin formation reaction is one of the important processes from both analytical and bioinorganic points of view.The large molar absorption coefficient and very high stability of the metalloporphyrins are very useful for the highly sensitive analysis of trace amounts of metal ions, and the size selectivity of the porphyrins is valuable for the separation of various kinds of metal ions. 1-7 A variety of metalloporphyrin formation rates are also applicable for the kinetic analysis of metal ions. We succeeded in the detection of the sitting-atop (SAT) copper(II) complex of TPP (5,10,15,20-tetraphenylporphyrin) in acetonitrile (AN) as a solvent with a very low Brønsted basicity, where two pyrrolenine nitrogens in the Cu(II)-SAT complex coordinate to the metal ion and two protons still remain on the pyrrole nitrogens. The structure parameters around the copper(II) ion in the Cu(II)-SAT complex, as determined by a fluorescent EXAFS method, suggest an axially elongated and equatorially distorted six-coordinate geometry. We measured the rates of the formation reaction of the SAT complexes for a series of transition metal(II) ions in AN using the stopped-flow technique. We propose the mechanism where there is a rapid deformation equilibrium of the porphyrin ring prior to the rate-determining step of the bond rupture of a coordinated solvent molecule on the metal(II) ion. Furthermore, we measured the rates of the deprotonation reaction of the Cu(II)-SAT complex by some Brønsted bases and indicated that the rate-determining step is the attack of the base on the proton of the pyrrole nitrogen in the SAT complex. Finally, a unified mechanism relevant to the porphyrin metalation mechanism has been proposed.

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“…Models have been proposed on the basis of kinetic studies, substrate specificity, and chemical modifications of certain amino acid residues and from analysis of the inhibition by N-alkylporphyrins (1)(2)(3)(4)16). The active site is believed to be an enclosed hydrophobic pocket (17), with cysteinyl (18) and arginyl (19) residues implicated in the binding of metal and porphyrin propionate(s). But the fact that no cysteine is conserved in the known ferrochelatase sequences makes it very unlikely that cysteine is involved in metal binding.…”

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Probing the Active-site Residues in Saccharomyces cerevisiae Ferrochelatase by Directed Mutagenesis

Góra

Grzybowska

Rytka

et al. 1996

Journal of Biological Chemistry

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Ferrochelatase is a mitochondrial inner membranebound enzyme that catalyzes the insertion of ferrous iron into protoporphyrin, the terminal step in protoheme biosynthesis. The functional/structural roles of 10 invariant amino acid residues were investigated by sitedirected mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase. The mutant enzymes were expressed in a yeast strain lacking the ferrochelatase gene HEM15 and in Escherichia coli. The kinetic parameters of the mutant enzymes were determined for the enzymes associated with the yeast membranes and the enzymes in the bacterial soluble fraction. They were compared with the in vivo functioning of the mutant enzymes. The main conclusions are the following. Glu-314 is critical for catalysis, and we suggest that it is the base responsible for abstracting the N-pyrrole proton(s). His-235 is essential for metal binding. Asp-246 and Tyr-248 are also involved in metal binding in a synergistic manner. The K m for protoporphyrin was also increased in the H235L, D246A, and Y248L mutants, suggesting that the binding sites of the two substrates are not independent of each other. The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had 1.2-2-fold increased V m and 4 -10-fold increased K m values for protoporphyrin, but the amount of heme made in vivo was 10 -100% of the normal value. These mutations probably affected the geometry of the active center, resulting in improper positioning of protoporphyrin.

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[47] Purification and characterization of mammalian and chicken ferrochelatase

Cited by 43 publications

References 13 publications

Molecular defects in ferrochelatase in patients with protoporphyria requiring liver transplantation.

Molecular defects in ferrochelatase in patients with protoporphyria requiring liver transplantation.

Dynamic Study of Metal-Ion Incorporation into Porphyrins Based on the Dynamic Characterization of Metal Ions and on Sitting-Atop Complex Formation

Probing the Active-site Residues in Saccharomyces cerevisiae Ferrochelatase by Directed Mutagenesis

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